Emergence and persistent spread of carbapenemase-producing Klebsiella pneumoniae high-risk clones in Greek hospitals, 2013 to 2022

Background Preliminary unpublished results of the survey of carbapenem- and/or colistin-resistant Enterobacterales (CCRE survey) showed the expansion of carbapenemase-producing Klebsiella pneumoniae (CPKP) sequence type (ST) 39 in 12 of 15 participating Greek hospitals in 2019. Aim We conducted a rapid survey to determine the extent of spread of CPKP high-risk clones in Greek hospitals in 2022 and compare the distribution of circulating CPKP clones in these hospitals since 2013. Methods We analysed whole genome sequences and epidemiological data of 310 K. pneumoniae isolates that were carbapenem-resistant or ‘susceptible, increased exposure’ from Greek hospitals that participated in the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE, 2013–2014), in the CCRE survey (2019) and in a national follow-up survey (2022) including, for the latter, an estimation of transmission events. Results Five K. pneumoniae STs including ST258/512 (n = 101 isolates), ST11 (n = 93), ST39 (n = 56), ST147 (n = 21) and ST323 (n = 13) accounted for more than 90% of CPKP isolates in the dataset. While ST11, ST147 and ST258/512 have been detected in participating hospitals since 2013 and 2014, KPC-2-producing ST39 and ST323 emerged in 2019 and 2022, respectively. Based on the defined genetic relatedness cut-off, 44 within-hospital transmission events were identified in the 2022 survey dataset, with 12 of 15 participating hospitals having at least one within-hospital transmission event. Conclusion The recent emergence and rapid spread of new high-risk K. pneumoniae clones in the Greek healthcare system related to within-hospital transmission is of concern and highlights the need for molecular surveillance and enhanced infection prevention and control measures.


Introduction
Klebsiella pneumoniae is a frequent cause of healthcare-associated infections including pneumonia, urinary tract, wound and bloodstream infections [1].Carbapenem-resistant K. pneumoniae infections are associated with a high mortality [2], primarily due to delayed administration of effective treatment and limited treatment options.Carbapenem resistance in Enterobacterales including K. pneumoniae has been recognised as a notable concern for patients and healthcare systems in countries of the European Union/ European Economic Area (EU/EEA) [3].In addition, the transmission of high-risk clones of carbapenem-resistant K. pneumoniae in healthcare settings has been identified as the major cause of the spread in EU/EEA countries [4].
National whole-genome sequencing (WGS)-based molecular surveillance for the detection and control of these clones is at various stages of implementation [5].European-level genomic surveillance of carbapenem-resistant K. pneumoniae high-risk clones has so far been based on structured multi-country surveys with central WGS and analysis.The first such survey was the European survey of carbapenemaseproducing Enterobacteriaceae (EuSCAPE) which collected isolates and data in 36 countries in 2013 and 2014 [4,6].It was followed by the survey of carbapenem-resistant and/or colistin-resistant Enterobacterales (CCRE survey) which collected isolates and data in 36 countries in 2019 [7].
Preliminary unpublished results of the CCRE survey showed the expansion of a new clade of carbapenemase-producing K. pneumoniae (CPKP) sequence type (ST) 39 carrying bla KPC-2 and/or bla VIM-1 in participating Greek hospitals.This clade was not present among isolates collected in 10 Greek hospitals in EuSCAPE in 2013 and 2014 [4] but was detected in 12 of 15 Greek hospitals participating in the CCRE survey in 2019 [7].
The CCRE survey results became available only after the COVID-19 pandemic in 2022.At this time, the extent of the spread of K. pneumoniae ST39 was not known and required further investigation to generate more timely data for infection prevention and control (IPC).
A national molecular follow-up survey was therefore conducted in 2022.Here we report the results of this follow-up survey which, combined with EuSCAPE and the CCRE survey data for Greece, allows tracking of high-risk clones in Greek hospitals over a 10-year period.

Data and isolate collection
The From July to September 2022, we used a modified CCRE survey protocol to conduct a molecular followup survey in 15 Greek hospitals that had participated in the CCRE survey [7].In short, the Greek National Reference Laboratory invited the hospitals to submit up to 10 consecutive K. pneumoniae isolates resistant to any carbapenem (based on European Committee on Antimicrobial Susceptibility Testing clinical breakpoints [10]) from individual patients accompanied by epidemiological and microbiological data.Phenotypic antimicrobial susceptibility testing (AST) of isolates was conducted at hospital microbiology laboratories with minimum inhibitory concentration (MIC) determination by VITEK 2 or disk diffusion for all analysed antibiotics except for colistin, which was evaluated by the commercial broth microdilution test available for routine use in each hospital.The epidemiological variables collected for this follow-up survey were the date of sampling, the healthcare institution submitting the sample, the sample type, the clinical relevance (infection or carriage), the status of the patient (inpatient or outpatient), the type of acquisition (community-or hospital-acquired), and age and sex of the patient.
Results from WGS and AST and epidemiological data of these isolates were combined with data on carbapenem-resistant (R) or carbapenem-'susceptible,

What did you want to address in this study?
A new highly drug-resistant clone of carbapenemase-producing Klebsiella pneumoniae had been detected in 12 of 15 Greek hospitals participating in a European genomic surveillance project in 2019.We looked at a collection of 10 carbapenem-resistant K. pneumoniae isolates per hospital to see how widely these bacteria had spread in 2022; such timely information could be used for infection prevention and control.

What have we learnt from this study?
The carbapenemase-producing K. pneumoniae detected in Greek hospitals over a 10-year period mainly belonged to a small number of so-called 'high-risk clones' known for their antimicrobial resistance and capacity to spread in healthcare settings.We also noted that new high-risk clones have recently emerged and spread rapidly throughout hospitals in the country.
What are the implications of your findings for public health?Infection prevention and control measures need to be enhanced immediately to prevent further spread of highly-drug resistant K. pneumoniae and similar resistant bacteria in hospitals.This study is a model for rapid national molecular surveillance studies that could also be applied in other countries and settings.

KEY PUBLIC HEALTH MESSAGE
increased exposure' (I) from Greek hospitals in the EuSCAPE and CCRE survey datasets.

Whole genome sequencing data analysis
Sequencing reads were assembled using SPAdes v3.15.3 [11] and analysed in Pathogenwatch [12].After quality control, 310 carbapenem-R/I isolates were included in this study: 40 from EuSCAPE (restricted to hospitals that also participated in the CCRE and followup surveys), 128 from the CCRE survey, and 142 from the follow-up survey.A neighbour-joining phylogenetic tree based on core genome single nucleotide polymorphisms (cgSNPs) was constructed using Pathogenwatch [12].In addition, STs (Institute Pasteur scheme) [13] and antibiotic resistance genes were determined using Kleborate v2.3.0 [14].
To estimate recent within-hospital transmission, we used the 2022 follow-up survey isolate pairs with ≤ 80 cgSNP pairwise distances determined in Pathogenwatch [12] within the same K. pneumoniae ST.Each pair was annotated as 'same hospital' or 'different hospitals' and cgSNP distances were calculated.We generated histograms showing distances for 'same hospital' and 'different hospitals' pairs.Transitions in the slope of the cumulative proportions per distance were used as signs of transition between phenomena and cluster cut-off candidates.

Participation and geographical representativeness
All 15 hospitals invited to participate in the follow-up survey sent isolates accompanied with epidemiological data.The number of isolates included in the final dataset ranged from five to 14 isolates per hospital with sampling dates between 22 February and 3 October 2022 (Table ).The 15 hospitals participating in the CCRE follow-up survey were located throughout the country, with 10 of 13 Greek NUTS-2 units represented with at least one hospital.For nine of these NUTS-2 units (EL43, EL51, EL52, EL53, EL54, EL61, EL62, EL63 and EL65), one hospital each was included, whereas for Attica (EL30), six hospitals were included taking into account that almost half of the Greek population lives in the main metropolitan region of Attica and also that this region receives many referrals for tertiary care from throughout the country.Combining all three surveys, this resulted in 128 carbapenem-R/I K. pneumoniae isolates in the dataset originating from EL30, while the other nine NUTS-2 units were covered with 13-30 isolates each (Table ).

Distribution of sequence types
Combining data of the three surveys showed that, in Greece, CPKP mainly belonged to five STs which Nomenclature of territorial units for statistics 2; I: susceptible, increased exposure; R: resistant.a The hospitals were selected based on their participation in the CCRE survey: 18 Greek hospitals had been invited to participate but only 15 responded and sent isolates.The three hospitals not responding to that survey, namely GR06, GR10, and GR16, are therefore not included in the dataset.b For this hospital, 14 isolates were included, deviating from the protocol requesting only up to 10 isolates per hospital.

Characteristics of Klebsiella pneumoniae ST39
All 56 ST39 isolates with predicted carbapenem resistance carried one single or several carbapenemase genes, including bla KPC-2 (alone or in combination with bla VIM-1 or bla NDM-1 ), bla KPC-3 , bla VIM-1 or bla VIM-4 , as well as mutations of genes encoding for major outer membrane porins, i.e.OmpK35 (amino acid sequence truncation) and OmpK36 (Gly115-Asp116 insertion).In addition, these isolates were also predicted to be fluoroquinolone-resistant since chromosomal mutations in the genes encoding for GyrA (S83I and D87N) and for ParC (S80I) were detected.Of these, 47 isolates were predicted to be colistin-resistant due to truncation in the MgrB regulator alone (n = 46) or in combination with truncation in PmrB (n = 1).Of these 47 isolates, 46 were harbouring genes for aminoglycoside-modifying enzymes among which more than half were aac(6')-Ib, aadA2, aac(3)-IId, aadA and/or aph(3)-Ia.For predicted virulence, most ST39 isolates (n = 46) carried the siderophore yersiniabactin gene ybt14 mobilised by the K. pneumoniae integrative conjugative element ICEKp5.

Within-hospital transmission
The optimum cut-off for 'same hospital'/'different hospitals' isolate pairs was determined to be between 7 and 11 cgSNP (Figure 5).To use a more conservative estimate, the cut-off for recent within-hospital transmission events was set to ≤ 8 cgSNPs and resulting pairs were evaluated for the most likely transmission pathway.Using this cut-off, 44 within-hospital transmission events were identified in the 2022 follow-up survey dataset, with 12 of 15 participating hospitals having at least one within-hospital transmission event.The number of within-hospital transmission events per hospital ranged from one to six and included 12 clusters of more than one related transmission event.Of these clusters, six were associated with ST11, two with ST258/512,  two with ST39, one with ST323 and one with ST147.All within-hospital transmission events involved K. pneumoniae high-risk clones, most frequently ST11 (n = 22 transmission events), followed by ST258/512 and ST39 (n = 7 each), ST323 (n = 6) and ST147 (n = 2).

Discussion
The worldwide spread of CPKP is driven by the transmission of international high-risk clones in healthcare facilities [4,16].This study documents the dissemination of high-risk clones of CPKP in the participating Greek hospitals over a 10-year period and shows the persistent spread of previously established as well as newly emerging high-risk clones.Three of the frequent K. pneumoniae STs in this dataset, ST258/512, ST11 and ST147, are known international high-risk clones also dominating in other EU/EEA countries and worldwide [4,16,17].Klebsiella pneumoniae ST258/512 and ST11 were widely distributed in all participating Greek hospitals and persistently detected over 10 years.While K. pneumoniae ST258/512 was the most frequent ST among CPKP isolates in Greece in 2009 and 2010 [18] and dominated in ECDC surveys in 2013 and 2014 and in 2019, K. pneumoniae ST11 was the most frequent in 2022.It is so far unclear whether CPKP ST11 has a competitive advantage over ST258/512 and if this trend will continue.Nevertheless, the large number of within-hospital transmission events associated with ST11 suggests that, once introduced in a hospital, this clone has a high potential for spread.An advantage of ST11 may also be its frequent association with bla NDM-1 which confers resistance to ceftazidime/avibactam in a context of increasing use of this combination antibiotic [19].
In Greece, highly drug-resistant K. pneumoniae ST39 carrying bla KPC-2 and bla VIM-1 , were recently reported in isolates from 2018 and 2019 [20].Our study shows that K. pneumoniae ST39 carrying bla KPC-2 has already spread to all 15 participating Greek hospitals within a short period and can therefore be considered a high-risk clone.This clone has so far only rarely been described outside of Greece; however, unpublished data from the CCRE survey show five sporadic isolates in five other EU/EEA countries and further investigation of potential cross-border spread is ongoing.Import of K. pneumoniae ST39 carrying bla KPC-2 from Greece to Finland has also been described [21].Of note, development of resistance to ceftazidime/avibactam during treatment has previously been reported for this clone [21,22].Even in a country with long-standing endemicity for CPKP such as Greece, the emergence of new high-risk clones is relevant as this is the starting point for further spread of these clones which usually have additional antimicrobial resistance mechanisms and/or are better adapted to transmission in healthcare settings.Our results document the steps in the emergence and spread of CPKP ST39 as a high-risk clone, through acquisition of resistance genes (mainly bla KPC-2 but also bla VIM-1 ) and mutations of genes encoding outer membrane porins (i.e.OmpK35/OmpK36) in a setting with high antibiotic pressure, and within-hospital and between-hospitals transmission events in a national healthcare system.Finally, another K. pneumoniae ST harbouring bla KPC-2 , namely ST323, that was not detected in Greece in 2019, appears to have rapidly spread to six Greek hospitals in 2022, potentially repeating the rapid increase of K. pneumoniae ST39.However, the spread of ST323 has mainly involved hospitals in Attica, Peloponnese and Crete, but not yet the six participating hospitals in northern and central Greece.Estimation of withinhospital transmission shows that the persistent spread of previously established CPKP STs and the rapid spread of new CPKP STs can probably be attributed to frequent transmission events in hospitals.In addition, the high percentage of CPKP isolates associated with infections in this study highlights the relevance of CPKP spread for clinical care.Phenotypic AST results show high levels of multidrug resistance in CPKP isolates, reducing available treatment options.However, it is a limitation of this study that these isolates have not been tested for newer antimicrobial substances, e.g.meropenem/vaborbactam, imipenem/relebactam, and cefiderocol.
Whole genome sequencing is a major development, which contributes to surveillance and control of antimicrobial resistance through early detection of transmission events and outbreaks, tracking of transmission pathways and the characterisation of involved pathogens and resistance mechanisms [23,24].Greece has a national surveillance for healthcare-associated infections caused by multidrug-resistant Gramnegative organisms in public and private hospitals called PROKROUSTIS, mandatory since 2014.As part of this system, national IPC guidelines have been provided and continuously updated, including guidelines for patient isolation.While most hospitals also perform screening for Gram-negative multidrug-resistant organisms at admission to intensive care, reporting is not mandatory and detailed information on screening practices and related results is therefore not available at national level.
To enhance IPC in Greek hospitals, a variety of additional actions could be considered, including (i) strengthening of IPC committees in hospitals with administrative support, (ii) increasing the number of IPC nurses at national level, (iii) establishing training modules for IPC specialists at medical schools and enhancing training for all healthcare professionals on IPC and antimicrobial stewardship (AMS), (iv) implementing new protocols for prevention of central line-associated bloodstream infections and catheterassociated urinary tract infections and (v) upgrading the surveillance system with a national database capable of mining information directly from hospital and laboratory information systems.In addition, the mandatory electronic prescription system for medical doctors at primary care level and establishment of AMS committees at hospital level would improve antibiotic usage.

Conclusion
The recent emergence and spread of K. pneumoniae ST39 and ST323 carrying predominantly bla KPC-2 in Greece is not only relevant because of their highly drug-resistant profile, but also as an example of rapid spread of newly emerging antimicrobial resistance threats throughout a hospital network.Similarly, rapid interhospital and interregional spread has been documented for CPKP and other emerging antimicrobial resistance threats in other EU/EEA countries [25][26][27].This situation is of concern and highlights the need for molecular surveillance and enhanced IPC measures in hospitals in Greece and in other EU/EEA countries, and more generally increased efforts to control antimicrobial resistance in the EU/EEA and beyond.

Ethical statement
All data were anonymised and collected in accordance with the European Parliament and Council decisions on the epidemiological surveillance and control of communicable disease in the European Community.Ethical approval and informed consent were thus not required.

Funding statement
The

License, supplementary material and copyright
This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence.You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.
Any supplementary material referenced in the article can be found in the online version.
This article is copyright of the authors or their affiliated institutions, 2023.

Figure 1
Figure 1Core-genome single nucleotide polymorphism-based phylogenetic tree of carbapenem-R/I Klebsiella pneumoniae isolates collected in 15 hospitals participating in two ECDC surveys and a national follow-up survey, Greece, 2013-2022 (n = 310)

Figure 2
Figure 2Distribution of the five most frequent sequence types of carbapenem-R/I Klebsiella pneumoniae isolates collected in 15 hospitals participating in two ECDC surveys and a national follow-up survey, Greece, 2013-2022 (n = 310)

Figure 3
Figure 3 Distribution of the five most frequent sequence types of carbapenem-R/I Klebsiella pneumoniae isolates collected in 15 hospitals participating in the CCRE survey and a national follow-up survey, Greece, 2013-2022 (n = 270)

Table Carbapenem -
R/I Klebsiella pneumoniae isolates from 15 hospitals participating in two ECDC surveys and a national followup survey, Greece, 2013-2022 (n = 310) CCRE: carbapenem-and/or colistin-resistant Enterobacterales; ECDC: European Centre for Disease Prevention and Control; EuSCAPE: European survey of carbapenemase-producing Enterobacteriaceae; NP: not participating; NUTS-2: Distribution of carbapenemases by sequence type of carbapenem-R/I Klebsiella pneumoniae isolates collected in 15 hospitals participating in two ECDC surveys and a national follow-up survey, Greece, 2013-2022 (n = 310) : carbapenem-and/or colistin-resistant Enterobacterales; ECDC: European Centre for Disease Prevention and Control; EuSCAPE: European survey of carbapenemase-producing Enterobacteriaceae; ST: sequence type. CCRE isolate and epidemiological data collection for EuSCAPE and the CCRE survey were funded by ECDC through specific framework contracts (ECDC/2012/055 and ECDC/2017/021).Whole genome sequencing (WGS) for EuSCAPE and the CCREFigure 5 Comparison of 'same hospital'/'different hospitals' isolate pairs using Pathogenwatch core genome single-nucleotide polymorphisms (cgSNPs) survey was funded by the Centre for Genomic Pathogen Surveillance, UK.WGS for the 2022 follow-up survey was funded by ECDC through a service contract with Eurofins Genomics.